Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment Stephanie Constantina, Daniel Reynoldsa, Andrew Oha, Katherine Pizanoa, and Susan Wraya,1 aCellular and Developmental Neurobiology Section, National Institute of Neurological Disorders and Stroke (NINDS), NIH, Bethesda, MD 20892 Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved November 23, 2020 (received for review June 15, 2020) 2+ Fertility relies upon pulsatile release of gonadotropin-releasing rate (20). Under normal conditions, [Ca ]i oscillations are driven hormone (GnRH) that drives pulsatile luteinizing hormone secre- by bursts of action potentials (AP) (21, 22). Yet, AP are not 2+ tion. Kisspeptin (KP) neurons in the arcuate nucleus are at the necessary for the KP-evoked [Ca ]i response to occur, as it is center of the GnRH pulse generation and the steroid feedback driven by multiple effectors including transient receptor potential- control of GnRH secretion. However, KP evokes a long-lasting re- canonical channels (TRPC), voltage-gated calcium channels sponse in GnRH neurons that is hard to reconcile with periodic (VGCC), and inositol 1,4,5-trisphosphate receptors (InsP3R) (15, GnRH activity required to drive GnRH pulses. Using calcium imag- 16, 19, 23). Thus, the versatility of Kiss1r signaling pathway un- ing, we show that 1) the tetrodotoxin-insensitive calcium response derlies the functionality of KP projections along GnRH neuron evoked by KP relies upon the ongoing activity of canonical tran- processes (24), with KP locally applied on nerve terminals also 2+ sient receptor potential channels maintaining voltage-gated evoking a long-lasting increase in [Ca ]i (16). calcium channels in an activated state, 2) the duration of the While the long-lasting KP response is suitable to trigger the calcium response is determined by the rate of resynthesis of phos- preovulatory surge, it seems incompatible with the KNDy model phatidylinositol 4,5-bisphosphate (PIP2), and 3) nitric oxide termi- for tonic pulses. GnRH and LH pulses occur every ∼20 min in nates the calcium response by facilitating the resynthesis of PIP2 ovariectomized mice (25). Indeed, the KNDy model provides via the canonical pathway guanylyl cyclase/3′,5′-cyclic guanosine on-/off- signals for KNDy neurons, neurokinin B and dynorphin monophosphate/protein kinase G. In addition, our data indicate respectively, and an on-signal for GnRH neurons, KP. However, that exposure to nitric oxide after KP facilitates the calcium re- this model lacks an off-signal for GnRH neurons. KNDy neurons NEUROSCIENCE sponse to a subsequent KP application. This effect was replicated trigger GnRH/LH pulses via Kiss1r (26), but the “extinction” of using electrophysiology on GnRH neurons in acute brain slices. The KNDy neurons by dynorphin is not obligatory for the termina- interplay between KP and nitric oxide signaling provides a mech- tion of GnRH/LH pulses in rodents (27, 28), and dynorphin is anism for modulation of the refractory period of GnRH neurons lacking in human KP-neurokinin neurons (29). after KP exposure and places nitric oxide as an important compo- 2+ In fact, how the response in firing and [Ca ]i to KP relate to nent for tonic GnRH neuronal pulses. GnRH secretion is unknown. In acute coronal brain slices, the KP-evoked increase in firing at the cell body cannot be repeated fertility | GnRH | kisspeptin | pulse | nitric oxide (19, 30). In contrast, in acute sagittal brain slices, using fast scan cyclic voltammetry, the KP-evoked GnRH secretion from cells in onadotropin-releasing hormone (GnRH)-secreting neurons the preoptic area (POA) and fibers in the median eminence Gintegrate multiple physiological and environmental cues and (ME) can be triggered repeatedly (31). The difference in re- translate them into GnRH secretory patterns, to drive gonado- peatability at the cell body is puzzling. One explanation could be trophs, which in turn control the gonads. In both sexes, tonic GnRH pulses regulate gametogenesis and steroidogenesis via Significance luteinizing hormone (LH) and follicle-stimulating pulses. Fe- males require a GnRH surge to trigger LH surge and ovulation Hypothalamic neural networks control hormone secretion to [reviewed in (1, 2)]. However, disruption of the normal pulsatile maintain body function. For gonadotropin-releasing hormone GnRH secretion impairs fertility in both sexes (3). Although (GnRH) neurons, critical to fertility, release must be pulsatile. insufficient for optimal reproductive health (4), the direct action While it is clear that kisspeptin neurons trigger GnRH activity of kisspeptins (KP) on GnRH neurons, via the KP receptor and pulses, how GnRH neurons return to baseline activity be- – Kiss1r, is required for fertility (4 6). KP neurons play a critical fore the next stimulation is unknown. Here, we show 1) that regulatory role in the hypothalamic–pituitary–gonadal axis, in- the long-lasting response of GnRH neurons to kisspeptin depends cluding puberty onset (7–9), the preovulatory GnRH surge (5, 9, on the degradation of phosphatidylinositol 4,5-bisphosphate 10), and tonic GnRH pulses (6, 11). In rodents, two KP neuronal (PIP2) and its slow resynthesis keeping canonical transient re- subpopulations exist with distinct functions: the anteroventral ceptor potential channels opened and 2) a mechanism whereby periventricular nucleus (AVPV) subpopulation, linked to pu- nitric oxide facilitates PIP2 resynthesis and GnRH neuron recov- berty onset [reviewed in (12)] and preovulatory surge [reviewed ery from kisspeptin activation. Defining the overlap in kisspeptin in (13)], and the arcuate nucleus (ARC) subpopulation, also and nitric oxide signaling pathways may provide an avenue for known as Kisspeptin-Neurokinin B-Dynorphin (KNDy) neurons, fertility treatment. linked to tonic pulses (reviewed in ref. 14). Exogenous KP at the GnRH cell body evokes a long-lasting Author contributions: S.C. designed research; S.C., D.R., A.O., and K.P. performed re- 2+ search; S.C. analyzed data; and S.C. and S.W. wrote the paper. increase in intracellular calcium levels ([Ca ]i) (15), often 2+ The authors declare no competing interest. leading to the summation of individual oscillations into [Ca ]i plateaus (15, 16). This observation is in agreement with an in- This article is a PNAS Direct Submission. crease in electrical activity where GnRH neurons in acute slices Published under the PNAS license. go from burst firing to tonic firing after KP application (17–19). 1To whom correspondence may be addressed. Email: [email protected]. One could argue that this prolonged response is an artifact of the This article contains supporting information online at https://www.pnas.org/lookup/suppl/ exogenously applied KP. However, endogenously released KP by doi:10.1073/pnas.2012339118/-/DCSupplemental. AVPV stimulation also evokes a long-lasting increase in firing Published December 21, 2020. PNAS 2021 Vol. 118 No. 1 e2012339118 https://doi.org/10.1073/pnas.2012339118 | 1of11 Downloaded by guest on September 24, 2021 Fig. 1. The tetrodotoxin-insensitive calcium response to KP-10 in GnRH neurons. (A) High-magnification images of GnRH neurons in explants identified by their morphology (Left), recorded with calcium imaging (Middle), and confirmed by immunocytochemistry (Right). Arrows indicate the same neurons throughout the experiment. (Scale bar, 20 μm in all panels.) (B) A heat map showing changes in [Ca2+] levels in 49 GnRH neurons recorded simultaneously. Neurons were perfused for 5 min in a control medium, and then TTX was added for the rest of the recording. After 5 min in TTX, the cells received a 2-min application of KP-10. Optical density (arbitrary units) was measured, with cells showing an increase in calcium levels changing toward yellow. (C) Examples of 2+ traces showing changes in [Ca ] levels (ΔF/F0) in five individual GnRH neurons (gray) and the average in black, evoked by 10 nM (Top) or 100 nM (Bottom)KP. The time of the arrow heads in C correspond to the time of arrow heads in D.(D) Average trace for changes in [Ca2+] levels (percent) evoked by 10 nM (open circles, n = 3 explants) and 100 nM (filled circles, n = 7 explants; reference trace for subsequent experiments). Each trace was normalized to its maximum to assess the recovery time at 50% (red horizontal line) for each dose (red vertical lines). The arrow heads represent the recovery period during which drugs were applied in subsequent experiments (i.e., once the response to KP had fully developed). (E) A bar graph depicting the average time (+SEM) at 50% recovery for explants used in D. The dotted line represents the value used as the reference (30.0 ± 1.4 min) without perturbations (KP 100 nM) for subsequent experiments. The average time at 50% recovery between 10 nM and 100 nM KP was not significantly different (nonparametric Mann–Whitney U test). 2of11 | PNAS Constantin et al. https://doi.org/10.1073/pnas.2012339118 Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment Downloaded by guest on September 24, 2021 a technical consequence of brain slicing or conventional patch integrates these KP inputs (32) and the calcium response is ac- clamp. Another explanation could be a dissociation between firing tion potential–independent (16). At 7 to 14 d in vitro, ∼90% of 2+ and [Ca ]i during the second KP application. In fact, the com- primary GnRH neurons from explants increase intracellular 2+ mon feature of the KP-evoked increase in [Ca ]i and